Progressive Degeneration of Human Mesencephalic Neuron- Derived Cells Triggered by Dopamine-Dependent Oxidative Stress Is Dependent on the Mixed-Lineage Kinase Pathway
Julie Lotharius,1Jeppe Falsig,1,2Johan van Beek,1Sarah Payne,3Ralf Dringen,4Patrik Brundin,5and Marcel Leist1
1Department of Disease Biology, H. Lundbeck A/S, 2500 Valby, Denmark,2Institute of Neuropathology, University Hospital Zurich, CH-8091 Zurich, Switzerland,3Cellomics Europe, Old Amersham HP7 0UT, United Kingdom,4Faculty 2 (Biology/Chemistry), University of Bremen, D-28334 Bremen, Germany, and5Section for Neuronal Survival, Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund University, 221 84 Lund, Sweden
Models of Parkinson’s disease (PD) based on selective neuronal death have been used to study pathogenic mechanisms underlying nigral cell death and in some instances to develop symptomatic therapies. For validation of putative neuroprotectants, a model is desirable in which the events leading to neurodegeneration replicate those occurring in the disease. We developed a humanin vitromodel of PD based on the assumption that dysregulated cytoplasmic dopamine levels trigger cell loss in this disorder. Differentiated human mesencephalic neuron-derived cells were exposed to methamphetamine (METH) to promote cytoplasmic dopamine accumulation. In the presence of elevated iron concentrations, as observed in PD, increased cytosolic dopamine led to oxidative stress, c-Jun N-terminal kinase (JNK) pathway activation, neurite degeneration, and eventually apoptosis. We examined the role of the mixed-lineage kinases (MLKs) in this complex degenerative cascade by using the potent inhibitor 3,9-bis[(ethylthio)methyl]-K-252a (CEP1347). Inhibition of MLKs not only prevented FeCl2⫹/METH-induced JNK activation and apoptosis but also early events such as neurite degeneration and oxidative stress.
This broad neuroprotective action of CEP1347 was associated with increased expression of an oxidative stress-response modulator, activating transcription factor 4. As a functional consequence, transcription of the cystine/glutamate and glycine transporters, cellular cystine uptake and intracellular levels of the redox buffer glutathione were augmented. In conclusion, this new human model of parkin- sonian neurodegeneration has the potential to yield new insights into neurorestorative therapeutics and suggests that enhancement of cytoprotective mechanisms, in addition to blockade of apoptosis, may be essential for disease modulation.
Key words: in vitro; reactive oxygen species; SAPK; neurodegeneration; apoptosis; MLK
Introduction
Nigral dopaminergic neurons are under continuous oxidative stress because of the unstable nature of their neurotransmitter dopamine (Graham, 1978; Maker et al., 1981), the adverse effects of which are further enhanced by high concentrations of iron in the substantia nigra (SN) (for review, see Zecca et al., 2004).
Experimental studies using methamphetamine (METH) suggest that abnormal accumulation of cytoplasmic dopamine could play an important role in disease pathogenesis by inducing oxi- dative stress (for review, see Lotharius and Brundin, 2002). Fur- thermore, nigrostriatal degeneration in mice treated with METH or the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), whose active metabolite can also induce dopamine-dependent oxidative stress (Lotharius and O’Malley, 2000), has been associated with an increase in the
phosphorylation of members of the c-jun N-terminal kinase (JNK) pathway, including c-Jun, JNK, and its upstream- activating kinase, mitogen-activated protein kinase kinase 4 (MKK4) (Saporito et al., 2000; Jayanthi et al., 2002; Willesen et al., 2002). In agreement with an active involvement of the JNK pathway in cell death, MPTP-induced neurodegeneration and motor deficits were significantly reduced in JNK2/JNK3 knock- out mice (Hunot et al., 2004), in mice overexpressing the inhib- itory JNK-interacting protein 1 (Xia et al., 2001), and in mice given the JNK inhibitor SP600125 (anthra[1,9-cd]pyrazol- 6(2H)-one) (W. Wang et al., 2004). Similarly, reduced c-Jun expression leads to increased resistance to METH-induced apo- ptosis (Deng et al., 2002).
Inhibition of the mixed-lineage kinases (MLKs), which acti- vate JNK indirectly via MKK4 and MKK7, can protect neurons from various insults. The potent MLK inhibitor 3,9- bis[(ethylthio)methyl]-K-252a (CEP1347) reduces JNK activa- tion and, consequently, neuronal death induced by trophic factor withdrawal (Maroney et al., 1999; Harris et al., 2002),-amyloid (Bozyczko-Coyne et al., 2001; Troy et al., 2001), axotomy (Glicksman et al., 1998), and MPTP exposure (Saporito et al., 1999). This is accomplished without inhibiting JNK directly, al-
We thank Trine Marie Nielsen and Andreas Rassov for excellent technical assistance, Tobias Minich for the GSH measurements, and Cephalon for CEP1347.
Correspondence should be addressed to Dr. Julie Lotharius, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark.
E-mail: mjl@lundbeck.com.
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lowing for physiological activation of JNK by other upstream kinases (Maroney et al., 1998, 1999, 2001; Bozyczko-Coyne et al., 2001; Troy et al., 2001; Harris et al., 2002). In addition to its antiapoptotic properties, CEP1347 exerts potent neurotrophic ef- fects in injured neurons, such as induction of neurite outgrowth, maintenance of neurotransmitter synthesis, and preservation of cel- lular metabolism (for review, see L. H. Wang et al., 2004).
To predict whether CEP1347 could be beneficial for treating neurodegenerative disorders such as Parkinson’s disease (PD), a broad range of experimental systems modeling disease pathogen- esis is desirable. Thus, we developed a new human cellular model of neurodegeneration based on the premise that cytosolic dopa- mine contributes to a loss of nigral neurons in PD. We exposed cultured human mesencephalic-derived cells (Lotharius et al., 2002) to METH and iron (FeCl2) to trigger endogenous dopamine-dependent oxidative stress and cell death. We further investigated whether CEP1347 could block neurotoxicity as well as early detrimental events involved in the collapse of neuronal function. Our data suggest that CEP1347 is not only antiapop- totic but also preserves neurite integrity by increasing cellular resistance to oxidative stress.
Materials and Methods
Chemicals
Methamphetamine was obtained from Lipomed (Arleshein, Switzer- land). Acridine orange (catalog #A3568) was from Molecular Probes (Eugene, OR); Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) was from OxisResearch (Portland, OR); 2-(4-morpholinyl)- 8-phenyl-4H-1-benzopyran-4-one (LY294002) was purchased from Calbiochem/EMD Biosciences (San Diego, CA); CEP1347 was provided by Cephalon (West Chester, PA). All other chemicals, including FeCl2 (catalog #F2130),N-acetyl-L-cysteine (NAC) (catalog #A9165), superox- ide dismutase (SOD) (from bovine erythrocytes; catalog #S5395), defer- oxamine mesylate (DM) (Desferal; catalog #D9533), catalase (from hu- man erythrocytes; catalog #C3556), and␣-methyl-L-tyrosine (␣-MPT) (catalog #M8131) were obtained from Sigma (Brøndby, Denmark) un- less otherwise specified. FeCl2was freshly prepared in double-distilled water for every experiment and used immediately.
Cell cultures
Lund human mesencephalic (LUHMES) cells are a subclone of the tetracycline-controlled, v-myc-overexpressing human mesencephalic- derived cell line MESC2.10, characterized at and originating from Lund University (Lund, Sweden) (Lotharius et al., 2002). LUHMES cells can be differentiated into morphologically and biochemically mature dopamine-like neurons after the addition of tetracycline, glial cell line- derived neurotrophic factor (GDNF), and db-cAMP and exhibit the same dopaminergic and neuronal characteristics as MESC2.10 cells [e.g., intense -III-tubulin immunoreactivity, extensive neuritic processes, time-dependent induction of tyrosine hydroxylase (TH), and extracellu- lar dopamine release], as measured by cyclic voltametry (data not shown). The culturing and handling procedure of LUHMES cells was as described previously (Lotharius et al., 2002), with the exception that all plasticware was precoated overnight with 50g/ml poly-L-ornithine (Sigma) at room temperature (RT) followed by a 3 h incubation with 1
g/ml fibronectin (Sigma) at 37°C to promote cell attachment. Plates/
dishes were washed three times with water and completely air dried before plating. All experiments were conducted after 4 d of differentia- tion in Ham’s F-12 high-glucose medium (Irvine Scientific, Santa Ana, CA) containing N2 supplement (Invitrogen, Glostrup, Denmark), 2 ng/ml human recombinant GDNF (R & D Systems, Wiesbaden- Nordenstadt, Germany), 1 mMdibutyryl cAMP (Sigma), and 1g/ml tetracycline (Sigma) after 4 d of differentiation. All experiments were vehicle controlled.
Primary ventral mesencephalic cultures
Primary rat ventral mesencephalic (VM) cultures were prepared as de- scribed previously (Boll et al., 2004). Briefly, VM tissue was dissected
from 14-d-old rat embryos and kept in ice-cold HBSS. The tissue was trypsinized in a solution containing 0.1% (w/v) trypsin and 0.05% (w/v) DNase dissolved in HBSS. After 20 min of incubation at 37°C, the tissue was rinsed four times in a 0.05% DNase–HBSS solution and mechani- cally dissociated. The cells were resuspended in DMEM containing 1 mM
sodium pyruvate, 15 mMHEPES, 1% penicillin/streptomycin, and 10%
(v/v) heat-inactivated fetal bovine serum and seeded on poly-L-lysine (50 mg/L)-coated 48-well plates at a density of 200,000 cells/well. Medium was replaced with Neurobasal medium supplemented with B27, 0.5 mm
L-glutamine, and 1% penicillin/streptomycin after 16 h, and one-half of the medium was replaced every other day with fresh Neurobasal me- dium. Cultures were pretreated with 100 –1000 nMCEP1347 and then exposed to 250MMETH for 3 d (dose responses and time courses were performed previously to establish toxicity of METH). Cultures were fixed and processed for TH immunocytochemistry. Experiments were con- ducted after 6 din vitro.
Cell viability assays
MTT reduction.Cells were plated in 48-well plates (Nunc, Roskilde, Den- mark) at a density of 45,000 cells/well and treated with Fe2⫹/METH in the presence or absence of various compounds for 72 h. To determine cell viability, the medium was removed, and cells were incubated with 5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro- mide (MTT) in fresh differentiation medium for 2 h at 37°C. At this point, the medium was removed, and cells were lysed in 95% isopropa- nol/5% formic acid under vigorous shaking for 15 min at RT. An aliquot was transferred to a 96-well plate, and the optical density was measured using a Multiscan Ascent spectrophotometer equipped with Ascent soft- ware version 2.4.2 (570 nm/690 nm filter pair; Titertek, Huntsville, AL).
Data are expressed as mean⫾SEM of quadruplicate determinations and are shown as a percentage of control cultures.
Lactate dehydrogenase release.The activity of lactate dehydrogenase (LDH) released into the medium was measured using the Cytotoxicity Detection kit (LDH) from Roche Diagnostics (Mannheim, Germany) according to the instructions of the manufacturer. Briefly, the entire medium from each well was transferred to an Eppendorf (Hamburg, Germany) tube and centrifuged for 2 min at 15,000⫻gto remove cellu- lar debris. A 50l aliquot of the supernatant was transferred to a 96-well plate, and 50l of freshly prepared reaction mixture was added. The plate was incubated for 25 min at RT while shaking, and the optical density was measured using the Multiscan Ascent spectrophotometer (492 nm/690 nm filter pair). Total LDH content was determined by lysing the cells in 0.3% Triton X-100 and measuring LDH in the lysate. Data are expressed as mean⫾SEM of quadruplicate determinations and shown as a per- centage of control cultures.
Calcein–AM staining.Membrane integrity was measured at the single- cell level using the LIVE/DEAD viability/cytotoxicity kit from Molecular Probes. Briefly, cells were incubated with 1Mcalcein AM for 20 min at 37°C and visualized by fluorescence microscopy. The number of calcein AM-positive cells was counted using the Target Activation BioApplica- tion in a Cellomics (Old Amersham, UK) ArrayScan high-content screening (HCS) automated microscope. Data are expressed as mean⫾ SEM of quadruplicate determinations and shown as a percentage of con- trol cultures.
Apoptosis. Assessment of nuclear condensation/fragmentation and plasma membrane integrity was conducted using the DNA-intercalating dye Hoechst 33342 and the membrane-impermeable marker SYTOX green (both from Molecular Probes). After drug exposure, LUHMES cells were incubated with 1g/ml Hoechst 33342 and 1MSYTOX green for 15 min at 37°C. Fluorescence was then visualized with a Zeiss (Oberkochen, Germany) Axiovert S100 TV microscope using excitation/
emission filter pairs 365/420 nm (Hoechst) and 450/565 nm (SYTOX green). Images were taken using MetaMorph software version 5.0 (Uni- versal Imaging, Downingtown, PA).
TH cell counting.The number of surviving dopaminergic neurons in ventral mesencephalic cultures was assessed by immunostaining cultures for TH using a 1:1000 dilution of monoclonal anti-TH antibody (catalog
#MAB318; Chemicon, Temecula, CA), the ABComplex/HRP detection system (Dako, High Wycombe, UK), and DAB as a chromogen. The
number of TH-positive neurons per well was counted by a blinded ex- aminer using a cast grid system. Data are expressed as mean⫾SEM of quadruplicate wells and shown as a percentage of control cultures.
Western blotting
Cells were lysed in 20 mMHEPES, 100 mMNaCl, 100 mMNaF, 1 mM
Na3VO4, 5 mMEDTA, 1% Triton X-100, and 1⫻complete protease inhibitors (Roche Diagnostics) for 15 min on ice. Protein determinations were conducted using a BCA Protein Assay kit from Pierce (Rockford, IL). Thirty micrograms of protein, heated for 10 min at 70°C, were elec- trophoresed on 4 –12% bis-Tris NuPage gels (Invitrogen) using 3-(N- morpholino)propanesulfonic acid SDS running buffer for⬃1.5 h at 200 V. Proteins were transferred onto an activated polyvinylidene difluoride membrane (Immobilon-P; Millipore, Glostrup, Denmark) using the XCell II blot module from Invitrogen. Membranes were blocked in 5%
milk in TBS/T (2.42 g/L Tris base, 8 g/L NaCl, and 0.1% Tween 20, pH 7.6), washed with TBS/T, and incubated overnight at 4°C with primary antibody dissolved in 5% milk in TBS/T. The following primary antibod- ies and their respective dilutions were used [all from Cell Signaling Tech- nology (Beverly, MA) unless otherwise specified]: 1:1000 polyclonal anti- phosphorylated AKT (Thr308), 1:2000 monoclonal anti--III-tubulin antibody (clone SDL.3D10; Sigma), 1:1000 polyclonal anti- phosphorylated c-Jun (Ser63), 1:100 polyclonal anti-cAMP response element-binding protein 2 [CREB2; activating transcription factor 4 (ATF4); Santa Cruz Biotechnology, Santa Cruz, CA], 1:500 monoclonal anti-phosphorylated JNK (Thr183/Thr185; clone G-7; Santa Cruz Bio- technology), 1:1000 polyclonal anti-phosphorylated MKK4 (Thr261), and 1:2000 monoclonal anti-glyceraldehyde-3-phosphate dehydroge- nase (GAPDH) antibody (clone 6C5; Novus Biologicals, Littleton, CO).
Blots were washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies [1:2000 rabbit anti-mouse and 1:1000 goat anti-rabbit from DakoCytomation (Glostrup, Den- mark)] for 1 h at RT and developed using ECL or ECL Plus detection reagents (Amersham Biosciences, Arlington Heights, IL).
pH measurements
Cells were plated at a density of 45,000 cells/well in 48-well plates and differentiated for 4 d. On day 4, cells were loaded with a 25 nMconcen- tration of the pH-sensitive dye acridine orange (catalog #A3568; Molec- ular Probes) and 0.2g/ml Hoechst for 40 min, followed by a 40 min incubation with 1 mMMETH. The ring-spot fluorescence intensity in the cytoplasm was measured using a Cellomics ArrayScan HCS automated mi- croscope (excitation/emission filter pair, 475/515 nm) using the Compart- mental Analysis BioApplication. All experiments were performed in quadruplicate.
Oxidative stress
Superoxide production.Cells were plated at a density of 200,000 cells/well in 12-well plates and differentiated for 4 d. On day 4, cells were exposed for 2– 60 h to FeCl2and METH in the presence or absence of CEP1347, loaded for 30 min at 37°C with 3Mdihydroethidium (which is oxidized to ethidium by superoxide anions), and lysed in 150l DMSO by freez- ing/thawing. The extract was transferred to a black 96-well plate, and fluorescence was measured with the Multiscan Ascent FL Fluoroscan (excitation/emission filter pair, 485/612 nm). Relative fluorescence units per sample were normalized to micrograms of protein. Protein determi- nations were conducted with a 5l aliquot from each sample using the BCA Protein Assay kit from Pierce.
Hydrogen peroxide formation.Cells were plated at a density of 400,000 cells per 5 cm dish, allowed to proliferate for 48 h, and differentiated for 4 d. Cells were exposed to METH in the presence or absence of CEP1347, rinsed with ice-cold PBS, and scraped off in 150l of PBS with a rubber policeman. FeCl2was omitted from this experiment because of interfer- ence of Fe2⫹with the assay reaction. Cells were homogenized by passing the resuspended cells 15 times through a 27 ga needle. The lysate was centrifuged at 1500⫻gfor 10 min, and a 50l aliquot of the supernatant was used to measure hydrogen peroxide (H2O2) using a commercial, colorimetric assay based on the oxidation of Fe2⫹to Fe3⫹by H2O2under acidic conditions (Bioxytech MDA-586; OxisResearch). An H2O2stan- dard curve was used to determine the absolute concentrations of H2O2in
the samples. Values were standardized to micrograms of protein as de- termined by a BCA Protein Assay kit from Pierce.
Lipid peroxidation.Cells were plated at a density of 400,000 cells per 5 cm dish, allowed to proliferate for 48 h, and differentiated for 4 d. After drug exposure, cells were rinsed with ice-cold PBS and scraped off in 250l of PBS containing 5 mMbutylated hydroxytoluene using a rubber policeman. Cells were homogenized by passing the lysate 15 times through a 27 ga needle and once through a 30 ga needle. The lysate was centrifuged at 1000⫻gfor 10 min, and a 200l aliquot of the superna- tant was used to measure malondialdehyde (MDA) levels using a kit from OxisResearch (Bioxytech MDA-586) according to the instructions of the manufacturer. This assay is based on the reaction of a chromogenic re- agent,N-methyl-2-phenylindole, with MDA at 45°C, and measures spe- cifically MDA and not other lipid peroxidation products. An MDA stan- dard curve was used to determine the absolute concentration of MDA in the samples using the stable MDA precursor tetramethoxypropane. Val- ues were standardized to micrograms of protein, as determined by a BCA Protein Assay kit from Pierce .
Glutathione measurements
For glutathione (GSH) measurements, LUHMES cells were plated at a density of 400,000 cells in 5 cm dishes and differentiated for 4 d. After drug exposure, LUHMES cells were rinsed twice with ice-cold PBS and lysed in 200l of 1% sulfosalicylic acid (w/v) on ice. The lysates were scraped off of the dish and centrifuged for 1 min at 15,000⫻g. Total glutathione content [reduced glutathione (GSH) plus oxidized glutathi- one (GSSG)] was determined in the supernatants using a microtiter plate assay as described previously (Dringen and Hamprecht, 1996), with a slight modification of the photometric method originally described by Tietze (1969). The pellet obtained from the centrifugation of samples was dissolved in 0.5MNaOH, and the protein content was determined by the method of Lowry. The amount of GSH per sample was standardized to the protein content of each respective precipitate. All samples contained
⬍1% GSSG, so that total GSH concentrations were almost identical to that of GSH in the cells.
Cystine uptake
A total of 80,000 cells/well were plated in 24-well plates and differentiated for 6 d. On day 6, cells were treated for 16 h with Fe2⫹/METH, 250 nM
CEP1347, or both. Cells were incubated for 20 min in 0.2Ci/ml [14C]cystine (9.25 GBq/mmol; PerkinElmer, Wellesley, MA) dissolved in prewarmed PBSG (10 mMPBS with 137 mMNaCl and 3 mMKCl, pH 7.4, containing 0.01% CaCl2, 0.01% MgCl2
䡠
6 H2O, and 0.1% glucose).Uptake was terminated by rapidly rinsing the cells three times in ice-cold PBS. Cells were then lysed in 0.5N NaOH by vigorous shaking. The lysate was transferred to a scintillation tube, and radioactivity was counted with a Packard Tri-Carb 2100TR counter (Global Medical Instrumentation, Ramsey, MN). MTT reduction assays were performed in parallel wells to ensure that Fe2⫹/METH or CEP1347 did not enhance cellular metabo- lism after the 16 h incubation period, which could possibly affect cystine uptake. Assays were run in six replicates.
Immunocytochemistry
Cells treated with 1 mMMETH or 75MFeCl2and 1 mMMETH in the presence or absence of 250 nMCEP1347 were fixed with 4% fresh para- formaldehyde for 20 min at 37°C and rinsed three times in PBS. Cells were blocked for 1 h with 5% goat serum in 0.3% Triton X-100/PBS and incubated overnight at 4°C with a 1:1000 dilution of a rabbit anti- mitogen-activated protein 2 (MAP2) antibody (catalog #AB5622;
Chemicon) or a 1:2000 dilution of a monoclonal anti--III-tubulin an- tibody (catalog #T0198; Sigma) dissolved in 0.3% Triton X-100/PBS.
Cells were rinsed three times in PBS and incubated for 1 h with 1:500 Alexa Fluor 488-coupled goat anti-mouse IgG (Molecular Probes) dis- solved in 0.3% Triton X-100/PBS. Cells were rinsed three times in PBS and visualized with a Zeiss Axiovert S100 TV microscope. In some cases, cells were incubated with 1g/ml Hoechst 33342 for 20 min in the second-to-last wash. Images were taken using MetaMorph software ver- sion 5.0.
Quantification of neurite degeneration
To quantify neurite degeneration resulting from Fe2⫹/METH exposure, a Cellomics ArrayScan HCS System was used. Cells were plated in 48-well plates at a density of 55,000 cells/well, differentiated for 4 d, and exposed to Fe2⫹/METH for 48 h. Cells were fixed with 4% fresh paraformalde- hyde and processed for-III-tubulin immunoreactivity using an Alexa Fluor 488-coupled secondary antibody and Hoechst 33342 (see above, Immunocytochemistry). Fluorescence was quantified using the GPCR (G-protein-coupled receptor) BioApplication from Cellomics, which can automatically subtract the fluorescence intensity of the cell body from the total fluorescence signal of the field being imaged to yield the neuritic intensity only. The size of the cell body was estimated using a modified mask (nuclear diameter plus 2M) of the nucleus of the neu- ron, which was defined by Hoechst 33342-positive pixels. Fifty fields/well were captured using a 10⫻lens. The “neurite mass” of these 50 fields was then averaged and normalized to that of control cultures. Each condition was run in quadruplicate.
Caspase-3 activation
Immunocytochemistry for activated caspase-3 was performed using an antibody specific for the cleaved protein [rabbit anti-human caspase-3 (Asp175) antibody; catalog #9661; Cell Signaling Technology]. Cells were incubated overnight with anti-activated caspase-3 antibody (1:500) at 4°C in a humid chamber. After washing, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500; cata- log #A11008; Molecular Probes) and 1g/ml Hoechst 33342 for 1 h at RT. Cells were visualized with an Axiovert S100 TV Zeiss microscope using excitation/emission filter pairs 365/458 nm (Hoechst) and 494/517 nm (Alexa Fluor 488).
Enzymatic activity was measured as described previously (Hentze et al., 2001). Briefly, cells were plated in 24-well plates at a density of 65,000 cells/well, differentiated for 4 d, and treated with CEP1347 for 6 –72 h.
Fe2⫹/METH was added at staggered time points, and cells were lysed at the same time with 80l of lysis buffer containing 20 mMHEPES, 5 mM
MgCl2, 1 mMEGTA, 0.4% Triton X-100, 4 mMDTT, and 1 mM4-(2- aminoethyl)benzenesulfonylfluoride, pH 7.4 (all from Sigma). Cells were frozen at⫺80°C and then thawed on ice to ensure lysis. The supernatant was removed from the wells and centrifuged at 15,000⫻gfor 2 min.
Subsequently, 50l of lysate was transferred to a black 96-well plate (Nunc), and the reaction was initiated by adding 50l of assay buffer containing 50 mM HEPES, 10 mM DTT, 1% sucrose, 0.1% 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 100M Ac-Asp-Glu-Val-Asp-7-Amino-4-trifluoromethylcoumarin (Ac-DEVD- AFC) substrate (Biomol, Plymouth Meeting, PA) to each well. The plate was shaken vigorously for 15 s, and fluorescence was measured with a Multiscan Ascent FL Fluoroscan using Ascent software version 2.4.2 [ex- citation/emission filter pair, 390/510 nm; kinetic interval, 20 ms; 90 scans (30 min)]. For analysis, the fluorescence increase between 10 and 20 min was measured and expressed as fluorescence units per minute. All values were normalized to those of control cells.
Quantitative PCR
Cells stimulated in 10 cm dishes were washed once with PBS, and total RNA was extracted using TRIzol reagent (Invitrogen) according to the protocol of the manufacturer. A DNase-free, DNase-1 kit was purchased through Ambion (Huntingdon, UK), and the purified RNA was treated according to the instructions of the manufacturer. Total RNA (1g) was used for additional analysis, as described previously (Falsig et al., 2004).
RNA was reverse transcribed with TaqMan reverse transcription Reagent (Applied Biosystems, Naerum, Denmark), using random hexamers in a 100l reaction mixture with a PTC-200 DNA Engine Thermal Cycler (VWR International, Albertslund, Denmark). The program consisted of 10 min of annealing at 25°C, 30 min of reverse transcription at 48°C, and 5 min of inactivation at 95°C. The cDNA was quantified using the SYBR Green PCR Master Mix kit (PE Applied Biosystems, Foster City, CA).
Each quantitative PCR (qPCR) contained 2.5l of cDNA of the 100l reverse transcription product, 300 nMforward and reverse primers, 12.5
l of master mix, and 7l of water in a total volume of 25l. PCR amplification was run in a 96-well experimental plate format on an iCy-
cler Thermal Cycler equipped with the iCycler Optical System (Bio-Rad, Hercules, CA). The program setup was 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 60°C. A melting curve was obtained to verify the measured signal, and the product was run on a 4% agarose gel to verify the presence of the expected band. Quantification was performed as follows: using the iCycler data analysis software (Bio-Rad), the threshold cycle (Tc) was determined for each sample. Tc was defined as the cycle at which the level of fluorescence increased significantly above the back- ground levels of fluorescence. The concentration of cDNA was calculated by comparing the Tc of samples to Tcs of a standard curve. The standard curve was obtained by a serial dilution of cDNA. Each sample was run in two reactions, one with the primer set of interest and one with a GAPDH primer set, and all data are displayed as the ratio between the calculated starting concentration of the cDNA of interest and GAPDH. All primers, except for the housekeeping gene GAPDH, were intron spanning to dis- tinguish cDNA from genomic DNA. For GAPDH, the order of magni- tude between the samples and the samples without reverse transcriptase was⬎106. Primers used were as follows: GAPDH sense (GenBank acces- sion number NM_008084), 5⬘-TCGACAGTCAGCCGCATCTTCTT-3⬘; antisense, 5⬘-GCGCCCAATACGACCAAATCC-3⬘; ATF4 sense (acces- sion number NM_009716), 5⬘-GTC TGC CCG TCC CAA ACC TTA C-3⬘; antisense, 5⬘-TCC TGC TCC GCC CTC TTC TTC-3⬘. Cystine– gluta- mate (Cys–Glu) transporter sense (accession number NM_014331), 5⬘- CCC TGG TCC GCC CTC TTC TTC-3⬘; antisense, 5⬘-TCG CAA GTT CAG GGA TTT CAC ATT-3⬘; glycine transporter (GlyT1) sense (accession num- ber NM_006934), 5⬘-AGG CCC TCA CAC TAC TTC CCA TCT C-3⬘; an- tisense, 5⬘-ACT CAT TCC CCA CCT CAT CCA CA-3⬘. Primers were de- signed using the DNAstar software package (DNASTAR, Madison, WI), and all primers were blasted using BLAST (basic local alignment search tool;
www.ncbi.nlm.nih.gov/BLAST/).
Statistics
Descriptive statistics (mean⫾SEM) of all quantitative assays performed were calculated with statistical software [GraphPad (San Diego, CA) Prism Software]. The mean of three to four individual experiments con- ducted in quadruplicate determinations is shown. The significance of effects between control cultures and Fe2⫹/METH or CEP1347 treat- ments was determined by one-way ANOVA andpost hocStudent’sttests (GraphPad Prism Software) (*p⬍0.05;**p⬍0.01; ***p⬍0.001).
Results
Progressive, dopamine-dependent cell death in differentiated LUHMES cultures by combined exposure to iron and methamphetamine (Fe2ⴙ/METH)
Amphetamines, including METH, lead to an accumulation of cytoplasmic dopamine (Sulzer et al., 1995; Mosharov et al., 2003), which can, in turn, promote oxidative stress (Cubells et al., 1994; Lotharius and O’Malley, 2001) and neurite degeneration in rodent neurons (Larsen et al., 2002). In the present study, we tested whether METH could be used as a tool compound to trig- ger dopamine-dependent oxidative stress in a human cell culture system. Differentiated LUHMES cells were exposed to 1 mM
METH for 1–5 d, and their dendritic morphology was assessed by MAP2 and-III-tubulin immunocytochemistry. After a 5 d in- cubation period, cells exhibited a dramatic loss of neurites, as shown by both-III-tubulin and MAP2 immunocytochemistry (Fig. 1A). LUHMES cultures treated with up to 1 mMMETH did not display a loss of cell viability, as assessed by a battery of cell toxicity/survival assays, including quantification of MTT reduc- tion capacity (an assay for mitochondrial dehydrogenase activ- ity), LDH release, ethidium homodimer uptake, and calcein AM retention, the latter three of which measure a loss in plasma membrane integrity (Fig. 1B). These data are in agreement with previous reports showing that a 7 d exposure to 100MMETH selectively destroys neurites but spares dopaminergic cell bodies in primary mesencephalic cultures (Cubells et al., 1994; Larsen et al., 2002). The inability of METH to induce dopaminergic cell
lossin vitrocould indicate an inherent capacity of dopaminergic neurons to combat dopamine-dependent oxidative stress, i.e., the threshold necessary to trigger apoptosis is not reached. Alter- natively, because the majority of intracellular dopamine is stored in synaptic vesicles (Sulzer et al., 1996), oxidative stress in this model could be restricted to the neurites, sparing the neuronal somata.
To tip the balance toward neuronal death, LUHMES cells were cotreated with METH (increasing cytosolic dopamine) and nontoxic concentrations of iron to catalyze the formation of cy- totoxic hydroxyl radicals from hydrogen peroxide via the Fenton reaction. In the SN of PD patients, redox-active iron levels are increased by 30 –35% compared with age-matched controls (for review, see Zecca et al., 2004; Go¨tz et al., 2004). Neuromelanin, a dark pigment produced in catecholaminergic neurons of the hu- man SN and locus ceruleus, acts as a storage system for iron in dopaminergic neurons (Zecca et al., 2002) and has been found to associate with large amounts of iron in diseased nigral tissue (Jell- inger et al., 1992, 1993). Neuromelanin could potentially serve a neuroprotective function by sequestering free intracellular iron.
However, if the iron-buffering capacity of neuromelanin is ex- hausted, as could be the case in PD, increased leakage of iron into the cytoplasm could act synergistically with elevated cytoplasmic dopamine to promote oxidative stress. To study the potential toxic interaction between increased cytosolic dopamine levels and redox-active iron, we cotreated LUHMES cells with METH
and FeCl2and determined their combinatorial effect on neuronal toxicity. Exposure to 75MFeCl2was nontoxic on its own and had minimal effects on neurite integrity (Fig. 1A,C,D); however, cotreatment with 1 mMMETH led to complete neurite disinte- gration (Fig. 1A) and a dramatic loss in cell viability seen after 3 d of drug exposure (Fig. 1C,D).
To determine whether Fe2⫹/METH-induced cell death is de- pendent on cytosolic dopamine and iron-mediated oxidative stress, LUHMES cells were treated with either the iron chelator DM (250M) or the tyrosine hydroxylase inhibitor␣-MPT (100
M) in the presence of Fe2⫹/METH. After 3 d, cell viability was assessed by measuring both MTT reduction capacity and by counting the number of calcein AM-positive neurons. Both Des- feral and ␣-MPT completely protected LUHMES cells from Fe2⫹/METH-induced cell death (Fig. 1D), supporting the notion that the toxicity conferred by this insult is dependent on both iron and dopamine.
The mode of cell death triggered by Fe2ⴙ/METH
LUHMES cells exposed to Fe2⫹/METH exhibited widespread nuclear fragmentation, which first became evident after 12 h of drug exposure (supplemental Fig. 1, available at www.jneurosci.
org as supplemental material). At 12–36 h, cells with fragmented nuclei still retained their plasma membrane integrity, as shown by the absence of labeling with SYTOX green, a cell-impermeable nucleic acid stain. After 48 h, cells with fragmented nuclei began to stain positively for SYTOX green, and, after 60 and 72 h, all apoptotic cells displayed broken plasma membranes. In agree- ment with this pattern of nuclear fragmentation, LUHMES cells treated with Fe2⫹/METH were strongly immunoreactive for pro- cessed caspase-3. Activated caspase-3 was readily detectable after 24 h of Fe2⫹/METH exposure in neurons exhibiting a significant degree of nuclear fragmentation but still had an intact plasma membrane (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Activated caspase-3 immunoreactiv- ity could be seen as late as 60 and 72 h after Fe2⫹/METH expo- sure, when cell bodies had become shrunken and neuritic pro- cesses had disintegrated. Caspase-3-like activity was measured enzymatically using the fluorogenic caspase-3, caspase-6, caspase-7, caspase-8, and caspase-10 substrate Ac-DEVD-AFC.
After 6 h, no change in Ac-DEVD-AFC cleavage was observed;
however, between 12 and 60 h after Fe2⫹/METH treatment, cells displayed a twofold to threefold increase in caspase-3-like activ- ity. The increase in activated caspase-3 temporally correlated with the progression of nuclear fragmentation. Notably, the level of caspase-3-like activity induced by Fe2⫹/METH in this culture system was relatively moderate compared with staurosporine- and colchicine-treated cells, which displayed a 16- and 24-fold increase in caspase-3-like activity, respectively, after 24 h of treat- ment (data not shown). To examine the causal contribution of caspases to Fe2⫹/METH-induced cell death, the pan-caspase in- hibitorN-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) was used. Although zVAD-fmk effectively blocked nuclear fragmentation assessed 48 h after Fe2⫹/METH treat- ment, it did not protect cells from Fe2⫹/METH-induced cell death when used at 10 –100M (data not shown). Although Fe2⫹/METH induced classical apoptotic changes, the slow nature of the insult, the mild activation of caspase-3, and the inability of caspase inhibitors to inhibit neuronal death suggest that this is not a classical apoptosis model.
Figure 1. Iron (FeCl2) significantly potentiates METH toxicity in differentiated LUHMES cells.
A, Differentiated LUHMES cells were treated with 1 mMMETH or 75MFeCl2alone or FeCl2plus METH (Fe/METH) for 5 d, and neurite integrity was qualitatively assessed by both-III-tubulin and MAP2 immunocytochemistry.B, Cell viability was measured in cultures exposed to 50 – 1000MMETH for 5 d by a battery of cell death/viability assays.C, Potentiation of METH toxicity was examined in cultures treated with 75MFeCl2, 1 mMMETH, or both (Fe/METH). Cell survival was assessed after 72 h by visual inspection of calcein AM-positive cells.D, A set of Fe2⫹/METH- treated cells (F/M) was cotreated for 72 h with 250MDM or 100M␣-MPT, and cell viability was quantified by counting the number of calcein AM-positive cells. All data are expressed as the mean⫾SEM of quadruplicate determinations and are shown as a percentage of control cultures. Error bars represent SEM.
The kinetics of neurite degeneration versus cell death
As mentioned above, treatment with METH alone induced neurite degenera- tion but not cell death. In LUHMES cells treated with FeCl2 in combination with METH, neurite beading and disintegra- tion were clearly evident before any changes in MTT reduction or LDH release could be seen, i.e., after 24 – 48 h (Fig. 2A).
FeCl2had only a minimal effect on den- dritic morphology, as determined by MAP2 staining, but induced a loss in over- all neuritic density, as shown by -III- tubulin immunocytochemistry (Fig. 1A).
After 24 h of Fe2⫹/METH exposure, LU- HMES cells began to exhibit significant beading of neuronal processes, which ulti- mately culminated in extensive neurite disintegration, detected by an overall de- crease in-III-tubulin and MAP2-labeled structures (Figs. 1A, 2A). Because in our cultures, -III-tubulin appeared to label axons and also dendrites in⬃50% of neu- rons, as opposed to MAP2, which labeled primarily dendritic structures and neuro- nal perikaya, we opted to use-III-tubulin as a general neuritic marker for quantifica- tion of degeneration of neuronal pro- cesses. LUHMES cultures treated with Fe2⫹/METH were immunoprocessed for
-III-tubulin, and an imaging algorithm was applied to selectively capture neuritic
-III-tubulin immunofluorescence (cell body staining was automatically ex- cluded), referred to as the neurite mass of the cultures (see Materials and Methods).
In agreement with visual assessments, cells exposed to Fe2⫹/METH for 24 –72 h exhibited a gradual, significant decrease in neuritic-III-tubulin fluorescence. After 24 h of exposure, cells already demon- strated a 21% decrease in neurite mass (Fig. 2B) 2 d before changes in MTT re- duction, LDH release, or calcein AM- positive cells could be detected (data not shown). Neurite degeneration was progres- sive, with LUHMES cells showing a gradual 45– 65% reduction in their neurite mass be- tween 48 – 60 h of Fe2⫹/METH exposure,
still with no evident decrease in the number of surviving cells (Fig.
2B). This assay therefore proved to be a more sensitive indicator of neuronal damage than traditional viability assays such as MTT re- duction, calcein AM staining, or LDH release and showed that Fe2⫹/ METH targets neurites days before it can damage neuronal somata.
JNK pathway activation and its blockade by the MLK inhibitor CEP1347
In addition to its well established role in the execution of apopto- sis (Lin and Dibling, 2002), recent evidence suggests that the stress-induced JNK pathway could be involved in the neurode- generative cascade leading to cell loss in PD (for review, see Sa- porito et al., 2002; Peng and Andersen, 2003). LUHMES cells
exposed to Fe2⫹/METH exhibited an increase in the phosphory- lated forms of various kinases belonging to the JNK signaling pathway. Increased levels of the phosphorylated JNK-activating kinase MKK4 were already seen 6 h after Fe2⫹/METH exposure and peaked after 36 h of treatment (Fig. 3A); changes in MKK7 phosphorylation were not observed (data not shown). An in- crease in Fe2⫹/METH-induced JNK phosphorylation was de- tected 24 h after Fe2⫹/METH exposure,⬃6 h after the onset of MKK4 activation, reaching peak levels after 36 h. Basal levels of phosphorylated c-Jun in LUHMES cells were high, but an addi- tional induction in phosphorylation was seen after 36 h of Fe2⫹/ METH exposure. We examined the possible role of MLKs as upstream activators of Fe2⫹/METH-induced toxicity by using Figure 2. Fe2⫹/METH leads to progressive neurite degeneration, which is significantly attenuated by CEP1347.A, LUHMES cells were treated with Fe2⫹/METH for 24 –72 h, fixed, and immunoprocessed for-III-tubulin. Cells were also cotreated with Fe2⫹/METH in the presence of 250 nMCEP1347 for 72 h.B, Neurite mass and cell viability were quantified in cultures immuno- stained for-III-tubulin 0 –72 h after exposure to Fe2⫹/METH, using a Cellomics ArrayScan instrument. Neuritic-III-tubulin immunofluorescence was used as a measure of neurite mass. Number of cells, as assessed by neurons with normal nuclear morphology, was used as an indicator of cell survival. Data are expressed as mean⫾SEM of quadruplicate determinations and are shown as a percentage of control.C, LUHMES cells were exposed to Fe2⫹/METH for 72 h in the presence or absence of 250 nM
CEP1347, and neurite mass was quantified with the Cellomics ArrayScan. Values are expressed as mean⫾SEM of quadruplicate determinations and are shown as a percentage of control.D, LUHMES cultures were treated with 250 nMCEP1347 (CEP) 6 – 48 h after Fe2⫹/METH (F/M) addition. All cultures were fixed and immunostained for-III-tubulin 72 h after the initial insult. Error bars represent SEM.
the potent MLK inhibitor CEP1347. Fe2⫹/METH-induced MKK4, JNK, and c-Jun phosphorylation was significantly re- duced after 36 h of exposure by cotreatment with 250 nM CEP1347 (Fig. 3B), suggesting a role for MLK in the activation of the JNK signaling pathway in this model.
Attenuation of Fe2ⴙ/METH-induced neurite degeneration, caspase activation, and cell death by the MLK
inhibitor CEP1347
Because CEP1347 blocked JNK pathway activation in cells treated with Fe2⫹/METH, we assessed the ability of the MLK inhibitor to block cell death and/or attenuate earlier pathogenic events pre- ceding the loss of cell viability. LUHMES cells were preincubated with CEP1347 for 1 h before the addition of Fe2⫹/METH. Via- bility was assessed by measuring LDH release into the media and MTT reduction capacity and by counting the number of calcein AM-positive cells after 72 h of Fe2⫹/METH exposure. CEP1347 completely prevented Fe2⫹/METH-induced cell death with an EC50of⬃100 nMaccording to all measurement parameters ex- amined (only MTT reduction and calcein AM-positive cell counts are shown) (Fig. 4A). The neuroprotective effect of CEP1347 did not appear to be transient, because robust neuro- protection was still seen 5 d after the initial insult. To confirm that the neuroprotective properties of CEP1347 were not restricted to this cell culture system, we examined the ability of the MLK in-
hibitor to block METH-induced toxicity in primary rat ventral mesencephalic cultures, which contain⬃5% dopaminergic neu- rons. These neurons, which were identified by TH immunocyto- chemistry, were, to our surprise, vulnerable to METH such that cotreatment with FeCl2was not necessary. A 3 d exposure to 250
MMETH led to a 50% decrease in TH-positive neurons, which could be completely rescued by pretreatment with CEP1347, with an EC50⬍100 nM(Fig. 4C).
Because a primary aim of antiparkinsonian medications would be to halt disease progression after the onset of neurode- generation, we examined whether CEP1347 could rescue LU- HMES cells if given hours to days after the initiation of the cell death cascade. LUHMES cells were exposed to Fe2⫹/METH 2– 48 h before the addition of CEP1347, and viability was assayed 72 h after the addition of the death-inducing stimulus by measuring Figure 3. Fe2⫹/METH induces activation of the JNK signaling pathway, which is blocked by
CEP1347.A, LUHMES cells were exposed to Fe2⫹/METH for 6 – 60 h, and the induction of JNK pathway intermediates was assessed by Western blotting using antibodies recognizing the phosphorylated forms of MKK4 (p-MKK4), JNK (p-JNK), and c-Jun (p-c-Jun). GAPDH was used as a loading control.B, LUHMES cells were cotreated with Fe2⫹/METH and 250 nMCEP1347 for 36 h, and the levels of p-MKK4, p-JNK, and p-c-Jun were measured by Western blotting. Blots shown represent three independent experiments.
Figure 4. The MLK inhibitor CEP1347 rescues LUHMES cells and primary rat ventral mesen- cephalic dopaminergic neurons from Fe2⫹/METH-induced toxicity.A, LUHMES cells were treated with Fe2⫹/METH for 72 h in the presence of 50 –1000MCEP1347, and cell viability was assessed by counting the number of calcein AM-positive cells and by measuring MTT re- duction capacity.B, Micrographs showing cultures treated with Fe2⫹/METH in the presence or absence of 250 nMCEP1347 stained with calcein AM.C, Primary rat ventral mesencephalic cultures were treated with METH alone or in the presence of 100 –1000MCEP1347, and dopaminergic cell survival was assessed by counting the number of TH-positive neurons. Values are expressed as mean⫾SEM of quadruplicate determinations and are shown as a percentage of control cultures.C, CEP1347 was added to LUHMES cells either 1 h before or 2–72 h after Fe2⫹/METH addition. The toxicity of Fe2⫹/METH was measured after 72 h by quantification of LDH release and expressed as a percentage of neuroprotection compared with control cultures.
All data are expressed as the mean⫾SEM of quadruplicate determinations.D, Caspase-3-like enzyme activity in LUHMES cells treated with Fe2⫹/METH in the presence or absence of 250 nM
CEP1347 was quantified for the ability to cleave a fluorogenic tetrapeptide, Ac-DEVD-AFC, which is a substrate for caspase-3, caspase-6, caspase-7, caspase-8, and caspase-10, after 6 – 60 h of drug treatment. The fluorescence emitted by cleavage of the substrate was quanti- fied during a 20 min incubation period by kinetic fluorimetric measurements. Values were standardized to micrograms of protein, expressed as mean⫾SEM of quadruplicate determi- nations, and shown as a percentage of control cultures. Error bars represent SEM.
LDH release. A minimum effective con- centration of CEP1347 (250 nM) rescued the majority of LUHMES cells from Fe2⫹/ METH-induced cell death, even when given as late as 24 h after initial exposure to the insult (Fig. 4D). This suggests that MLK inhibitors can halt the progression of an ongoing pathogenic process. Notably, coincubation of Fe2⫹/METH-treated cells with 250 nMCEP1347 completely blocked nuclear fragmentation (supplemental Fig.
1, available at www.jneurosci.org as sup- plemental material), assessed after 72 h of drug exposure, as well as caspase-3-like ac- tivation (Fig. 4E), measured after 6 – 60 h.
As mentioned above, LUHMES cells exposed to Fe2⫹/METH exhibited sig- nificant neurite beading and disintegra- tion. This early event was completely blocked by 250 nMCEP1347, as assessed by visual inspection of the cultures 72 h after cotreatment with the MLK inhibi- tor (Fig. 2A). In agreement with qualita- tive assessments of CEP1347-mediated neurite rescue, CEP1347 significantly at- tenuated Fe2⫹/METH-induced neuritic dystrophy in a novel quantitative neurite degeneration assay. The total neurite mass in cells cotreated with CEP1347 and Fe2⫹/METH was 270% higher than in cells treated with Fe2⫹/METH alone (Fig. 2C). CEP1347 retained its ability to prevent neurite degeneration even when added 12–24 h after Fe2⫹/METH expo- sure (Fig. 2D). Although extensive bead-
ing could be seen in some cells given CEP1347 24 h after Fe2⫹/METH, the majority of neurites remained intact. Never- theless, these results suggest that the commitment for neurite loss lies somewhere between 12–24 h and clearly demonstrate that CEP1347 is not only neuroprotective but also prevents the occurrence of earlier dystrophic changes such as neurite degenera- tion in human mesencephalic-derived cells.
No evidence for involvement of AKT activation in the neuroprotective effects of CEP1347
A recent study by Roux et al. (2002) showed that CEP1347 could promote the activation of AKT, a prosurvival kinase, in primary cortical neurons, an effect most likely independent of its inhibi- tory effect on the MLKs. To determine whether CEP1347 blocked Fe2⫹/METH-induced neurite degeneration and cell death by ac- tivating AKT, protein levels of Thr308phosphorylated AKT (p- AKT) were measured in LUHMES cells treated with Fe2⫹/METH in the presence or absence of CEP1347. Although Fe2⫹/METH treatment alone did not change protein levels of p-AKT mea- sured after 6, 24, and 36 h (a transient 50% decrease was observed only at the 12 h time point), cotreatment with CEP1347 led to a persistent upregulation of the activated form of the kinase first detected after 6 h of drug addition (Fig. 5A). This increase in p-AKT expression induced by Fe2⫹/METH and CEP1347 was blocked by treatment with 10MLY294002 (Fig. 5B), suggesting that upregulation of p-AKT by CEP1347 involves the phospho- inositide 3-kinase (PI3K) pathway.
To determine whether activation of the PI3K–AKT pathway
was involved in the neuroprotective effects of CEP1347, cells were cotreated for 72 h with Fe2⫹/METH and CEP1347 in the presence or absence of the PI3 kinase inhibitor LY294002 (10
M), and cell survival was assayed by the MTT reduction assay.
Fe2⫹/METH treatment led to a 60% decrease in cell viability, which was completely blocked by CEP1347 (Fig. 5C). The PI3K inhibitor alone decreased cell viability by⬃25% and significantly potentiated the degree of cell death induced by Fe2⫹/METH.
However, CEP1347 retained its ability to reduce cell death in the presence of LY294002, suggesting that the PI3K pathway is not necessary for the ability of CEP1347 to rescue cells from Fe2⫹/ METH. One flaw of the experiment is the relatively low stability of LY294002 (t1/2⬍12 h). Given the late onset of AKT activation (⬎6 h), LY294022 may not have adequately blocked PI3K at later time points. Therefore, LY294002 was added in a parallel set of experiments several hours after combined Fe2⫹/METH and CEP1347 addition to ensure that PI3K was adequately inhibited.
Even when added 6 – 60 h after Fe2⫹/METH and CEP1347 addi- tion, LY294002 did not decrease the ability of CEP1347 to rescue cells from Fe2⫹/METH-induced toxicity as measured by LDH release into the media (Fig. 5D). These results suggest that, al- though AKT is activated in response to CEP1347, it is not in- volved in the rescue of Fe2⫹/METH-induced toxicity.
Blockade of Fe2ⴙ/METH-induced oxidative stress by CEP1347
METH is known to induce dopamine-dependent oxidative stress in mesencephalic neurons (Cubells et al., 1994; Larsen et al., Figure 5. Activation of AKT and its role in the neuroprotective effects of CEP1347.A, Protein levels of phosphorylated AKT (Thr308) were measured by Western blotting in whole-cell lysates from LUHMES cultures treated with Fe2⫹/METH for 6 –36 h in the presence or absence of 250 nMCEP1347.B, Levels of p-AKT were measured by Western blotting after 24 h of treatment with CEP1347, Fe2⫹/METH with CEP1347 (F/M⫹CEP), or Fe2⫹/METH with CEP1347 in the presence or absence of a 10Mconcen- tration of the PI3 kinase inhibitor LY294002. ForAandB,-actin was used as a loading control. Blots shown are representative of three independent experiments, and numbers under the blots correspond to densitometric quantification of band densities.C, LUHMES cells were treated with Fe2⫹/METH in the presence or absence of 250 nMCEP1347 and 10MLY294002. MTT reduction was used to assess cell survival after 72 h. Values are expressed as mean⫾SEM of quadruplicate determinations and are shown as a percentage of control.D, To exclude the possibility that LY294002 may be inefficacious in blocking PI3K at later time points during⬎6 h of CEP1347 exposure, the inhibitor was added 6 – 60 h after the addition of Fe2⫹/METH and CEP1347. Viability was assayed after 72 h by measuring the release of LDH into the medium. Values are expressed as mean⫾SEM of quadruplicate determinations and are shown as a percentage of control. Error bars represent SEM.
